LUCO Viral RNA Extraction Kit

Catalogue No.: KD474883-500 (500 reactions)

Catalogue No.: KD474883-50 (50 reactions)

Advanced Molecular Diagnostics Ltd is a diagnostics company specialising in

the manufacture and supply of molecular biology instruments, reagents and
consumables.

For use in In Vitro Diagnostics.

info@am-diagnostics.co.uk
+44 (0) 115 969 9934
Address:
Biocity Nottingham, Pennyfoot Street, Nottingham, NG1 1GF, United Kingdom

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Contents

Kit Contents ................................................................................................... 3

Overview ....................................................................................................... 4
Storage Specifications .................................................................................... 4
Basic Principles .............................................................................................. 4
Protocol ......................................................................................................... 5
Sample Collection: .......................................................................................................................... 5
Sample Preparation:....................................................................................................................... 5
Stage 1: Lysis and binding .............................................................................................................. 6
Stage 2: Washing ............................................................................................................................ 7
Stage 3: Elution ............................................................................................................................... 7

Protocol Summary ......................................................................................... 8

Additional Information .................................................................................. 9

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Kit Contents

Item KD474883-50 KD474883-500

VLB buffer 1 x 30ml 1 x 300 ml
VW1 buffer 1 x 16.5 ml 1 x 165 ml
VW2 buffer 1 x 10 ml 1 x 100 ml
Elution buffer 2 x 2 ml 1 x 40 ml
Carrier RNA 160 μg 1.6 mg

VLB buffer * - lysis buffer

Carrier RNA – increases RNA yield by binding to nucleic acids and aiding with
precipitation.

VW1 buffer* – removes a large proportion of contaminants e.g., proteins,

inhibitors etc. *100% ethanol must be added before extraction* (see bottle
label for volume)

VW2 buffer* – This reagent includes a mix of ethanol and water to remove salts
from the RNA, increasing the purity of the final sample. *100% ethanol must
be added before extraction*(see bottle label for volume)

Elution buffer – RNase free water

*
Contains chaotropic salts

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Overview

The LUCO Viral RNA Extraction Kit provides a time-efficient and simple method
to extract viral RNA from a variety of sample types e.g. plasma, serum or any
other bodily fluid. This product will allow the user to extract viral RNA with ease,
speed and precision using silica-based membrane technology using spin column.

The protocol has been designed to maximise RNA yield and purity, whilst
minimising degradation and removing inhibitors, reducing problems in
downstream applications. The extracted nucleic acid can be used in various
downstream applications.

Storage Specifications

The kits should be transported and stored at temperatures between 15 °C and

30 °C. The kit will remain stable at least until the expiry date printed on the
package if the storage temperature is kept.

Basic Principles

AMD Viral RNA Extraction Kit is designed for rapid and efficient extraction of
high-quality viral RNA from various human liquid samples such as plasma,
serum, whole blood, saliva, nasal and buccal swabs, urine and urogenital swabs.
The kit utilizes a silica-based membrane technology in the form of a convenient
spin column.

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Protocol

Sample Collection: The sample for AMD Viral RNA Extraction Kit can be
collected from a variety of sources, as described above. Please ensure that the
sample is stored correctly and kept away from any contamination.
* Nasopharyngeal Swab (NP) /Oropharyngeal Swab (OP): If you get a
swab without transport media, place the swab with into Microcentrifuge
tube containing PBS and incubate for 15 minutes at Room Temperature.
Afterwards shake the swab vigorously, squeeze it against the wall of
tube and remove the swab.

Sample Preparation: Please see the protocol below for details of the AMD Viral
RNA Extraction kit. Before starting the protocol:

1. VW1 and VW2 buffers require 100% ethanol adding to the solution,
before extractions take place.
2. Resuspend the supplied lyophilized vial of Carrier RNA of supplied RNase-
free Water (elution buffer) at a concentration of 1 μg/μl and mix
thoroughly.
3. Prepare VLB Buffer containing resuspended Carrier RNA; on average
about 560 μL of VLB Buffer is prepared for each RNA isolation (i.e., for 10
samples, add 5.6 ml VLB buffer to 56 μl resuspended Carrier RNA).

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 *Carefully design the expected number of isolations you are going to
do per week and make aliquots of resuspended Carrier RNA accordingly
and store them at -80 °C for up to 6 months. Carrier RNA enhances
binding of Viral RNA to the silica membrane and reduces the risk of Viral
RNA degradation, but Carrier RNA has a limited shelf-life of 4 weeks in
VLB Buffer when stored at 4 °C. In addition, more than 3 freeze-thaw
cycles of Carrier RNA MUST be avoided.

Stage 1: Lysis and binding

1. Allow all reagents and samples to equilibrate at room temperature.

2. In a nuclease free eppendorf, add 560 μl of the VLB buffer

(containing Carrier RNA) to 140 μl of the sample.

3. Vortex the sample thoroughly and incubate at room temperature

for 10 minutes.

4. Add 560 μl of ethanol (96-100%) to the sample and mix by vortex

5. Set up the spin column/tube and transfer 630 μl of the lysed

sample. Centrifuge at 14000g for 1 minute. Discard the flow-through.

6. Repeat step 5 for the rest of the sample until it is all collected to the
Min-spin column.

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Stage 2: Washing

7. Transfer the Mini-spin column to a new collection tube and add 500
μl of VW1 buffer to and centrifuge at 14000g for 1 minute. Discard the
flow through.
8. Place the Mini-spin column to the same collection tube and add
500μl of VW2 buffer and centrifuge again at full speed for 1.5 minutes.
Discard the flow through. Repeat this step twice.

Stage 3: Elution

9. Before elution, centrifuge the spin column for 2-3 minutes at

16000g without adding any solutions to the column
10. Remove the spin column from the original collection tube and
replace with a fresh nuclease free eppendorf.
11. Add 60 μl RNase-free water to the spin column directly in the
middle of the filter. Optional step: incubate the spin column with the
Elution Buffer for up to 2 minutes to increase yield.
12. Elute the newly extracted DNA from the spin column by
centrifuging for 14000g for 1 minute
13. Store the newly extracted DNA in -200C until required for further
experiments

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Protocol Summary

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Additional Information

Sample volume requirement – We recommend using 140 μl of the original

sample, to produce an adequate and high-quality RNA yield.

Elution volume – Depending on your required final RNA concentration, please

add 50-100 μl of elusion buffer.

Additional equipment Required (not provided):

Up to 200 μl of sample: Any of the following samples can be used with this
AMD kit; plasma, serum, whole blood, saliva, nasal and buccal swabs, urine
and urogenital swabs.

Disposables: AMD manufacture high quality PCR plastic wear suitable for
use with this kit. It is also recommended that correct PPE including gloves is
used throughout.

Other Laboratory Equipment: Vortex, centrifuge, pipettes and tips.

Sample storage: Generally, multiple freeze-thaws should be avoided. The most

practical way to address this concern is by aliquoting samples after collection.

Sample transport: Sample material should be transported in a shatterproof

transport container as a matter of principle. Thus, a potential danger of infection
due to a leakage of sample can be avoided. The samples should be transported
following the local and national instructions for the transport of pathogen
material. The samples should be shipped within 6 hours. It is not recommended

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to store the samples where they have been collected. It is possible to ship the
samples by mail, following the legal instructions for the transport of pathogen
material. We recommend the sample transport with a courier.

Warnings and precautions: When working with chemicals, always wear a

suitable lab coat, disposable gloves, and protective goggles. For more
information, please consult the appropriate safety data sheets (SDSs). Discard
sample and assay waste according to your local safety regulations.

Product Use Limitations: All reagents may exclusively be used in in vitro

diagnostics. The product is to be used by personnel specially instructed and
trained in the in vitro diagnostics procedures only. Strict compliance with the
user manual is required for optimal PCR results. Attention should be paid to
expiration dates printed on the box and labels of all components. Do not use
expired components.

Contact: Any queries, comments or complaints please refer to our website

Advanced Molecular Diagnostics Ltd.

info@am-diagnostics.co.uk
Tel. +44 (0) 115 969 9934
Biocity Nottingham, Pennyfoot Street, Nottingham
NG1 1GF, United Kingdom

www.am-diagnostics.co.uk

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